Agricultural biotechnology has progressed significantly since the 1980s, allowing development of a number of improved genetically modified crop plants which demonstrate resistance to pests and improved tolerance to commercial herbicides. However, currently available model plant systems for screening transgenes are inefficient, requiring extensive time for generating transformants. In vitro model systems, such as hairy roots, may also exhibit variability which renders results difficult to interpret. In addition, a phenotype observed in a model plant or plant tissue transformed with a transgene may not be observed in a crop plant transformed with the same transgene. The drawbacks of existing model systems may be attributable in part to existing transformation methodology and in part to the nature of the plant or plant tissue being transformed.
WO 00/12735 discloses an assay for screening transgenes for functional activity that employs a stable chimeric plant having wild-type shoots, stems, and leaves, but having transgenic “hairy roots” (neoplastic outgrowths of fine roots caused by the soil bacterium Agrobacterium rhizogenes). In the assay of WO 00/12735, an explant of the plant species to be assayed is wounded, and the wounded portion is infected with A. rhizogenes that is “armed”, i.e., capable of causing disease. The A. rhizogenes contains a transgene that may be incorporated into 40-90% of the hairy roots that form below the wounding site. Tissues above the wounding site are not infected by the A. rhizogenes and thus do not contain the transgene.
Commonly assigned WO 2006/024509 discloses “disarmed” strain variants of A. rhizogenes strain K599 useful for transforming plants to generate whole transgenic plants with normal (non-hairy) root phenotype. In the transformation methods of WO 2006/024509, after co-cultivation with A. rhizogenes strain K599, plant shoots are induced prior to root induction, and the ultimate transformed plant which is obtained is transgenic in all tissues.
Agricultural pests and climate change have negatively influenced humanity's ability to grow enough food to feed itself. In addition, the need to replace fossil fuels with more sustainable energy sources has made development of biofuels an important goal of the 21st century. A large number of transgenes must therefore be screened for functionality in crop plants in order to solve existing agricultural challenges. A need exists for improved methods to facilitate the ease, speed and efficiency of high throughput transformation systems for evaluating expression of transgenes that mediate a variety of root-specific phenotypic traits, including disease resistance, insect resistance, herbicide tolerance, abiotic stress tolerance, and the like.